18 research outputs found
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Retinoic acid inhibits NFATc1 expression and osteoclast differentiation
Ingestion of excess vitamin A appears to correlate with an increased fracture risk, an outcome that is likely mediated by retinoic acids (RAs); these are vitamin A metabolites that have dramatic effects on skeletal development. We studied the impacts of RA and isoform-specific RA receptor (RAR) agonists (α, β, and γ) on osteoclast formation (osteoclastogenesis) in two model systems: RAW264.7 cells and murine bone marrow-derived monocytes. The pan-RAR agonists, all-trans and 9-cis RA, inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclast differentiation in a concentration-dependent manner. Isoform-specific RAR agonists (α, β, and γ) also inhibited osteoclastogenesis, with the RARα agonist producing the most consistent reductions in both osteoclast number and size and total area covered. Inhibition of osteoclastogenesis correlated with reductions in expression, DNA binding, and nuclear abundance of nuclear factor of activated T cells c1 (NFATc1), a transcription factor critical for osteoclastogenesis. The upregulation of three NFATc1-responsive genes, cathepsin K, dendritic cell-specific transmembrane protein and osteoclast-associated receptor were similarly reduced following RA or RAR agonist exposure. These results suggest that RA blocks in vitro RANKL-mediated osteoclastogenesis by decreasing NFATc1 function
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AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells
Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption, and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by a complex physiological network of hormones and cytokines. A critical regulator of this process is receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of cytokines that can act via the TNF receptor activating factor (TRAF6)/AP-1 signaling pathway. However, the mechanism whereby RANKL modulates CTSK expression is not fully understood. Therefore, we investigated the regulation of CTSK expression and promoter activity in RAW 264.7 osteoclast precursor cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Western blot analysis, real-time RT-PCR and luciferase reporter gene assays revealed that RANKL stimulated CTSK expression and promoter activity in a dose- and time-dependent manner and that this activation was inhibited by either dominant negative (DN) TRAF6 or DN-c-fos. TRAF6 stimulated the basal activity of a truncated CTSK promoter, and DN-c-fos blocked this stimulation. JunB alone also stimulated basal CTSK promoter activity, whereas c-jun, JunD or c-fos alone did not. However, co-transfection of any of these jun-family members with c-fos (AP-1) significantly increased CTSK promoter expression. siRNA targeted against c-jun or junB suppressed RANKL-mediated CTSK expression. Therefore, both TRAF6 and AP-1 help regulate the basal and RANKL-mediated stimulation of CTSK gene expression in RAW 264.7 cells
A clinically relevant CTLA4-Ig-based regimen induces chimerism and tolerance to heart grafts
Background. We determined whether a nontoxic CTLA4-Ig-based conditioning regimen effected mixed chimerism and donor-specific tolerance when heart and bone marrow were transplanted simultaneously.
Methods. Fully mismatched rat strain combinations were used. Recipients received total-body irradiation (300 centigrays), bone marrow (10
8 cells), and cardiac transplants from the donor on day 0. Subsequently, recipient animals received CTLA4-Ig (2 mg/kg, every other day, × 5 doses), tacrolimus (1 mg/kg/day; days 0 to 9), and one dose (10 mg) of antilymphocyte serum on day 10.
Results. All bone marrow recipients (n = 7) developed mixed chimerism (mean = 25% ± 9% at 1 year) and accepted cardiac allografts permanently (> 375 ± 32 days). Recipients that received conditioning regimen but no bone marrow (n = 5) rejected donor hearts within 51 ± 13 days (
p 180 days) donor-specific skin grafts, but rapidly rejected (< 10 days) third-party skin grafts.
Conclusions. A nontoxic CTLA4-Ig-based conditioning regimen effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously. This regimen may have clinical application
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Molecular dissection of mouse soluble guanylyl cyclase α1 promoter
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Combined host-conditioning with CTLA4-Ig, tacrolimus, anti-lymphocyte serum, and low-dose radiation leads to stable mixed hematopoietic chimerism
The toxic dose of irradiation required to achieve stable mixed hematopoietic chimerism is the major limitation to its clinical application in transplantation and other nonmalignant conditions such as hemoglobinopathies. This study examines the additive effect of costimulatory blockage, to our previously described tacrolimus-based conditioning regimen, in further reducing the dose of total-body irradiation to achieve stable mixed chimerism in rats.
Fully mismatched, 4- to 6-week-old ACI and Wistar Furth rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig 2mg/kg/day (alternate days) in combination with tacrolimus 1 mg/kg/day (daily) from day 0 through day +10, anti-lymphocyte serum 10 mg at day +10 (single dose), and total-body irradiation ranging from 100–600 cGy, prior to bone marrow transplantation (day 0) with 100 × 10
6 of T-cell-depleted bone marrow cells. Levels of donor chimerism were determined over a period of 12 months.
The short course of CTLA4-Ig, tacrolimus, and ALS led to dramatic engraftments at reduced doses of irradiation: 100% (5/5) and 93% (13/14) of the animals developed mixed chimerism at 400 cGy and 300 cGy, respectively. At 300 cGy, recipients exhibited durable, multilineage mixed chimerism at 365 days with donor cells ranging from 19–42% (mean 23.4%) with no evidence of graft-vs-host disease. These mixed chimeras exhibited in vitro (mixed lymphocyte reaction) and in vivo (skin grafts) donor-specific tolerance.
This study suggests that addition of costimulatory blockade to a tacrolimus-based conditioning regimen reduces the dose of irradiation required to achieve stable multilineage chimerism in rats
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Identification of NFAT binding sites that mediate stimulation of cathepsin K promoter activity by RANK ligand
The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL
COSTIMULATORY BLOCKAGE WITH CTLA4Ig SIGNIFICANTLY REDUCES THE DOSE OF TOTAL BODY RADIATION (200 cGy) TO ACHIEVE MIXED CHIMERISM IN RATS Abstract# 940 Poster Board #-Session: P197-III
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